P313: RECURRENT PATHOGENIC GERMLINE CHEK2 VARIANTS IN PEDIATRIC PATIENTS WITH HEMATOLOGICAL MALIGNANCIES
Authors: T. Brozou, R. Wagener, U. Fischer, A. Borkhardt
Journal: HemaSphere (2022)

Background:CHEK2 is a tumor-suppressor gene encoding CHEK2 kinase that plays a crucial role in DNA repair and cell cycle arrest as part of the ATM-CHEK2-p53 pathway. Predisposing germline variants in CHEK2 are associated with breast cancer and other adult-type malignancies such as prostate, gastric and thyroid cancer. Only few studies have examined the occurrence of germline CHEK2 variants in adults with leukemia, but the association between inherited CHEK2 variants and hematological malignancies in childhood has not been studied. Aims: Our aim is to understand the frequency and the impact of germline variants in the cancer predisposing CHEK2 gene in pediatric hematological malignancies. Methods: We analyzed parent-child WES data of 150 children (≤18yrs) with hematological malignancies (n=107 leukemia, n=43 lymphoma) and mined the data for CHEK2 variants. We performed functional analysis of the identified pathogenic variants to analyze the effect of the alteration on protein function. Results: We detected in 15/150 children with hematological malignancies a CHEK2 germline variant (Figure 1A). The majority of those patients had a leukemia (n=11 BCP-ALL, n=1 T-ALL, n=1 CML, n=2 Hodgkin Lymphoma). Overall, eight different CHEK2 variants including three truncating variants were detected. Three variants were recurrent (p.Ile157Thr n=5, p.Ile160Met n=3, p.Thr367Metfs*15 n=2). According to the classification of the American College of Medical Genetics and Genomics 7/8 variants were predicted to be pathogenic or likely pathogenic. For two of these variants an impaired function had been already described in the literature. To test the functional consequences of the five remaining variants, we overexpressed these, in comparison to two control CHEK2 variants, in U2OS cells. Four out of seven variants (n=1 truncating, n=1 in-frame deletion, n=2 missense) led to loss of protein expression (Figure 1B) and loss of CHEK2-Thr68 phosphorylation upon irradiation induced DNA damage. Five out of fifteen patients carried a CHEK2 variant impairing its function. Interestingly, 4/5 patients with a pathogenic variant had a BCP-ALL and 1/5 had a CML that rapidly progressed to CD10+, CD19+ lymphoid blast crisis while under imatinib therapy (400 mg/m2). Figure 1: (A) Schematic representation of the CHEK2 protein indicating the localization of the identified variants. The length of the bars correlates to the number of patients with the respective CHEK2 variant and the color of the bars to the variant type (green: missense, red: frameshift indels, orange: splicing). (B) Western blot analysis of HA-tagged CHEK2 protein variants ectopically expressed in U2OS cells. GAPDH was used as a loading control. CHEK2 variants Arg145Trp and Glu161 del were included as controls in our experiments. (C) Bar blot depicting the quantification of the CHEK2 variant protein expression analyzed by Western blotting of three independent experiments. Each experiment is represent by a dot, the bar indicates the mean of all three experiments. Image:Summary/Conclusion: We identified heterozygous CHEK2 variants in the germline of 10% of children with hematological malignancies by WES-based screening of 150 cases. Expression studies revealed that two of the variants identified in our cohort were associated with reduction/loss of CHEK2 expression. We conclude that reduced CHEK2 expression/phosphorylation upon DNA damage may be an important germline-encoded host factor predisposing to the development of BCP-ALL in children.